Method and use of nano-scale devices for reduction of tissue injury in ischemic and reperfusion injury

ABSTRACT

A method for protection of tissues subject to ischemic and/or reperfusion damage is provided. The method includes administering to the tissue a composition comprising nanodevices. The nanodevices can take the form of, for example, polymeric nanoparticles or lipidic nanoparticles. The nanodevices also find use in methods for reducing ischemic injury in tissue at risk of such injury, such as heart and brain tissue.

CROSS REFERENCE TO RELATED APPLICATIONS

The application is a divisional of U.S. application Ser. No. 11/799,573,filed May 1, 2007, now allowed, which claims the benefit of U.S.Provisional Application No. 60/796,790, filed May 1, 2006; each of whichis incorporated herein by reference in its entirety.

STATEMENT REGARDING GOVERNMENT INTEREST

This invention was made with Government support under contract HL052141awarded by the National Institutes of Health. The Government has certainrights in this invention.

TECHNICAL FIELD

The subject matter described herein relates to methods for reducingischemic and reperfusion-induced tissue injury by delivering nanodevicesto achieve a therapeutic benefit under conditions ofischemia/reperfusion.

BACKGROUND

Ischemic and reperfusion injury are major causes of disability and deathin the United States. Ischemia is caused by a blockage and cessation ofblood flow to a region of tissue, and may occur in multiple tissues,including the heart in diseases such as myocardial infarction and thebrain, such as in ischemic stroke. Reperfusion injury may occurfollowing recanalization of an occluded vessel, and reflow of blood intoan ischemic area. Reperfusion may cause additional tissue stress,sometimes worsening damage.

Current strategies to protect tissues from the effects of ischemic andreperfusion damage are limited. The primary approach involves thedelivery of drugs for prevention, such as anti-platelet agents (e.g.aspirin, abciximab), anti-coagulants (e.g. warfarin, tissue plasminogenactivator (tPA)), anti-inflammatory agents (e.g. aspirin), diuretics(e.g. furosemide), vasodilators (e.g. nitroglycerine, ACE inhibitors),and anti-hypertensive medications (e.g. atenolol). Such drugs reduce thecausative factors involved in arterial blockage; however, they do notprovide protection to a tissue affected by an ischemic event. Inaddition, not all patients benefit from these treatments, due in part tofactors including drug insensitivity, drug toxicities, and other risks(e.g. hemorrhage), and drug interactions.

In addition to drug-based strategies, approaches have been developed todiminish the injury associated with an acute ischemic event. Suchtechniques focus on restoring blood flow by use of angioplasty, arterialstenting, coronary bypass, and treatment with thrombolytic drugs (e.g.tPA). These treatments may improve patient prognosis, however, tissuedamage can occur from the procedure including the acute risk of vesselrupture and ischemic damage, and delayed risks include restenosis, orreocclusion of the occluded vessel leading to additional ischemicevents. For example, following arterial stenting procedures, restenosisoccurs in 10-40% of cases (van der Hoeven, B. L. et al., Int J.Cardiol., 99(1):9 2005).

There a need for a treatment method for reducing tissue damage in anischemic and reperfusion events. In addition, there exists a need fornanodevices for the treatment of patients, including patients in whichcurrent pharmaceutical solutions are unacceptable.

BRIEF SUMMARY

The following aspects and embodiments thereof described and illustratedbelow are meant to be exemplary and illustrative, not limiting in scope.

In one aspect, a method for reducing ischemic reperfusion injury isprovided by delivering intravascularly a composition comprisednanodevices. Delivery of such nanodevices is beneficial in methods fortissue protection and for reducing tissue damage subsequent to anischemic event.

In another aspect, methods for protecting cardiac tissue in a mammalianheart prone to, characterized by, or otherwise experiencing, ischemicinjury due to a disease or condition, such as myocardial infarction, areprovided. In one embodiment, a method for decreasing tissue damage insuch a mammalian heart includes administering to a patient in needthereof an effective concentration of a nanodevice. Nanodevices includenanoparticles, nanorods, nanospheres, microspheres, liposomes, micelles,microbubbles, or nanobubbles. In certain embodiments, the agent is apolymer nanoparticle, and is further described as poly(methylidenemalonate 2.1.2) (PMM 2.1.2). More generally, and in another embodiment,the nanodevice is comprised of a polymer, including biodegradablepolymers, such as poly(lactic-co-glycolic acid). Other polymers that mayadvantageously be used in the methods are further described below

The methods may advantageously be used to decrease ischemic and/orreperfusion injury in conditions characterized by ischemic damage. Themethods may also be utilized to decrease ischemic damage in multipleother tissues in vivo (such as the brain, kidney, or liver) for anyother purpose where tissue protection against ischemic damage isdesired, including but not limited to, during organ and tissue harvestand transplantation and during surgeries in which blood supply to theorgan is temporarily interrupted.

In another aspect, methods for delivery of agents which confer tissueprotection against a timed (or scheduled) ischemic event (e.g., surgery)or untimed ischemic event (e.g., myocardial infarction or stroke) areprovided. In one embodiment, a patient is provided with an effectiveconcentration of a nanodevice within a clinically-relevant time-frameprior to the surgery. In one embodiment, this time frame is less than 48hours, preferably less than 24 hours, more preferably less than 12 hoursprior to surgery, still more preferably within about 6 hours prior tosurgery. Alternatively, the nanodevices are delivered chronically (i.e.,for an indefinite period of time) to reduce damage from an untimedischemic event, for example to a person at risk of myocardial infarctionor stroke. Chronic delivery may be every month, week, day, or hour.

In another aspect, methods for intra-vascular delivery of nanodevices toconfer a therapeutic benefit are provided. In one embodiment,nanodevices are delivered via a directly supplying artery or vein to thetarget tissue prior to an ischemic event. In various embodiments, thenanodevices are injected intravascularly, e.g., at peripheral sites forsystemic delivery to achieve a therapeutic benefit.

In other aspects, methods for decreasing tissue damage in a mammaliantissue characterized by, or otherwise experiencing, an ischemic orreperfusion event due to a disease or condition, such as myocardialinfarction, are provided.

In still other aspects, methods for reducing tissue damage in amammalian tissue prone to, characterized by, or otherwise experiencing,ischemic injury due to a disease or condition, such as myocardialinfarction, in patients that are unresponsive, desensitized, orotherwise unable to benefit from available drug agents, are provided.Such patients include those receiving drugs or not receiving drugs.

In related aspects, the use of a nanodevice for the preparation of amedicament for administration to a subject for protecting tissue fromischemic injury is provided. In some embodiments, the nanodevice is aplurality of nanodevices.

In some embodiments, the nanodevice is selected from the groupconsisting of nanoparticles, nanorods, nanospheres, liposomes, micelles,and nanobubbles.

In some embodiments, the nanodevice is a polymeric nanodevice. Inparticular embodiments, the polymer is a biodegradable ornonbiodegradable polymer.

In some embodiments, the polymer is poly(methylidene malonate 2.1.2)(PMM 2.1.2).

In some embodiments, the subject is at risk of myocardial infarction. Insome embodiments, the subject is at risk of ischemic damage due tomyocardial infarction or stroke. In some embodiments, the administeringto a subject is prior to surgery.

In another aspect, the use of a nanodevice for the preparation of amedicament for administration to a subject intravascularly for reducingischemic reperfusion injury to a tissue is provided.

In some embodiments, the medicament is administered during organ ortissue harvest. In some embodiments, the medicament is administeredduring organ or tissue transplantation. In some embodiments, themedicament is administered during surgery.

In some embodiments, the nanodevices are polymeric nanodevices. In someembodiments, the nanodevices are lipidic nanodevices.

In another aspect, the use of a nanodevice for the preparation of amedicament for administration to a subject intravascularly forconferring protection to a tissue at risk of ischemic injury, whereinthe nanodevice is a polymer or lipidic nanodevice, is provided.

In some embodiments, the medicament is administered prior to a scheduledischemic event. In some embodiments, the scheduled ischemic event issurgery. In particular embodiments, the medicament is administered atleast about 12 hours prior to surgery.

In some embodiments, the medicament is administered chronically prior toan unscheduled ischemic event. In particular embodiments, the medicamentis administered at least about monthly.

In some embodiments, the lipidic nanodevices are administered in theform of liposomes or micelles.

In all embodiments, the medicament may further comprise an additionaltherapeutic agent.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by reference to thedrawings and by study of the following descriptions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows images of heart slices obtained from animals exposed toischemic reperfusion injury with mock-treatment (control) or ischemicreperfusion injury with prior intravascular infusion of nanoparticles(PMM nanoparticles), with or without recirculation of perfusate buffer(i.e., recirculating, non-recirculating, respectively).

FIGS. 2A and 2B are graphs showing the average infarct sizes in heartssubject to ischemic-reperfusion injury with mock-treatment (control), orwith prior intravascular infusion of nanoparticles, with withoutrecirculation of the perfusate buffer (A), or with recirculation of theperfusate buffer (B). (*p<0.05—statistical significance between groupsassessed by students t-test).

FIG. 3 is a graph showing the average creatine phosphokinase levels inhearts subject to ischemic-reperfusion injury with mock-treatment, orwith prior intravascular infusion of nanoparticles, with our withoutrecirculation of the perfusate buffer (*p<0.05—statistical significancebetween groups assessed by students t-test).

DETAILED DESCRIPTION

In one aspect, methods of decreasing tissue damage in a vertebratetissue prone to, characterized by, or otherwise experiencing, ischemicinjury due to a disease or condition are provided. As used herein,“ischemia” refers to an event that causes a cell, tissue, or organ toreceive an inadequate supply of oxygen. It has been discovered thatintravascular delivery of nanodevices to an organ affected by ischemiaand/or reperfusion reduces tissue damage under conditions of ischemiaand reperfusion. In addition, it has been discovered that delivery ofnanodevices prior to an ischemic event significantly reduces damagecaused by a subsequent ischemia.

I. Nanodevices

In one embodiment, the present methods include administering to apatient in need thereof a therapeutically effective dose of nanodevices.As used herein, a “nanodevice” refers to a device that is of a sizebetween 1-10,000 nm, more preferably between 10-5,000 nm, and still morepreferably between 10-1,000 nm. The nanodevice may be of virtually anygeometry, including but not limited to nanoparticles, nanospheres,nanorods, and nanobubbles. An exemplary nanoparticle is a polymericnanoparticle. Another exemplary nanoparticle is a lipidic nanoparticle,such as a liposome or a micelle. These devices may have modifications asdescribed below to enhance the therapeutic effect, improve delivery,tissue targeting, biocompatibility, stability, pharmacokinetics,toxicity, or other benefits. The term nanoparticle may be used to refergenerally to the nanodevice but should not be construed as limiting.

In one embodiment, polymers may be used to form nanoparticles ormicroparticles. This polymer may a natural or synthetic polymer,hydrophilic or hydrophobic, biodegradeable or non-biodegradable.Exemplary biodegradable polymers include but are not limited topolyesters including polylactic acid) (PLA), poly (lactic-co-glycolicacid) (PLGA), poly(glycolic acid) (PGA), poly-e-caprolactone (PCL), orpolyanhydrides (e.g., bis(p-carboxyphonoxy) propane and sebacic acid).Natural polymers include but are not limited to gelatin, collagen,keratin, chitosan, alginate, and other natural polymers known in theart. Non-biodegradeable polymers include but are not limited tomethylcellulose, polyacrylarnide, poly-2-hydroxyethyl methacrylate,polyhydroxyethyl methacrylate (pHEMA), polymethylmethacrylate (PMMA),polyvinyl alcohol (PVA), and polyethylene glycol (PEG). Homopolymers maybe combined to form di and tri-block copolymers, and/or othercombinations thereof. A range of molecular weights from, for example, 1kDa to 1 megaDa, may be used. In general, a variety of polymers with arange of positive, negative, or neutral charges can be used, as in, forexample, co-polymers with various ratios of polymer components. Polymerscan be selected on the basis of several parameters including size,surface characteristics (including charge), biocompatibility, minimalcytotoxicity and immunogenicity, or other adverse side effects.

In another embodiment, lipids are used to form micellar or liposomalnanoparticles. Formation of micelles and liposomes from, for example,vesicle-forming lipids, is known in the art. Vesicle-forming lipidsrefer to lipids that spontaneously form lipid bilayers above theirgel-to-liquid crystalline phase transition temperature range. Suchlipids typically have certain features that permit spontaneous bilayerformation, such as close to identical cross-section areas of theirhydrophobic and hydrophilic portions permitting packing into lamellarphases. Lipids capable of stable incorporation into lipid bilayers, suchas cholesterol and its various analogs, can be incorporated into thelipid bilayer during bilayer formation. The vesicle-forming lipids arepreferably lipids having two hydrocarbon chains, typically acyl chains,and a head group, either polar or nonpolar. There are a variety ofsynthetic vesicle-forming lipids and naturally-occurring vesicle-forminglipids, including the phospholipids, such as phosphatidylcholine,phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol, andsphingomyelin, where the two-hydrocarbon chains are typically betweenabout 14-22 carbon atoms in length, and either saturated or havingvarying degrees of unsaturation. The above-described lipids andphospholipids whose acyl chains have varying degrees of saturation canbe obtained commercially or prepared according to published methods.Other suitable lipids include phospholipids, sphingolipids, glycolipids,and sterols, such as cholesterol.

Polymeric and lipidic nanodevices can additionally include a coating ofa hydrophilic polymer. For example, the nanodevices can include apolymer-polyethylene glycol or a lipid-polyethylene glycol conjugate, toprovide an external surface coating of polymer chains. The polymerpolyethylene glycol is exemplary, and other polymers are suitable andare contemplated. Preparation of a vesicle-forming lipid derivatizedwith a hydrophilic polymer to form a lipopolymer is described, forexample in U.S. Pat. No. 5,013,556. It will also be appreciated that thepolymer or lipidic nanodevice can be formed from multiple layers of thesame or different material.

A variety of nanodevice sizes may be used based on factors including theconcentration, route of delivery, target tissue and disease application.Sizes from about 1 to 10,000 nm, more preferably 1 to 1,000 nm, andstill more preferably 100 to 600 nm, are contemplated.

A wide variety of modifications to the nanodevices may be made and areknown in the art. Such modifications may include coating the nanodevicewith surfactants (e.g. PEGlyation), stabilizers (e.g. dextran, MW50,000-70,000), or agents that enhance delivery or targeting to tissues,such as targeting moieties, including but not limited to antibodies andantibody fragments with specific binding to a cell surface receptor.

The nanodevice may also be coated or impregnated with a therapeuticagent; however, it is appreciated that the nanodevice alone (e.g., withno coated or impregnated therapeutic agent) provide a therapeuticeffect. In some embodiments, addition of a therapeutic agent to thenanodevice, e.g., to provide an additional, different, or synergisticresponse, is contemplated. As used herein, a “therapeutic agent,” oradditional therapeutic agent,” refers to any molecule, e.g., protein,oligopeptide, small organic molecule, polysaccharide, polynucleotide,etc., which can be attached to a nanodevice for subsequent release. Suchtherapeutic agents include anti-platelet agents (e.g., aspirin,abciximab), anti-coagulants (e.g., warfarin, tPA), anti-inflammatoryagents (e.g., aspirin), diuretics (e.g., furosemide), vasodilators (e.g.nitroglycerine, ACE inhibitors) and anti-hypertensive agents (e.g.,atenolol). In one embodiment, a single drug is loaded or impregnatedinto the nanodevice. In another embodiment, multiple drugs are loaded orimpregnated into the device, e.g., for co-delivery to a patient. Suchtherapeutic agents may be used to enhance or provide additionaltherapeutic benefits.

The nanodevice may also contain or be coated with non-therapeutic agentsto enhance the delivery of drugs, protect the drugs from degradation orstabilize the drug during processing, storage, or patient use, orprotect the polymer material from adverse effects during processing,storage or patient use. In addition, the nanodevice may contain agentsthat reduce toxicity or adverse side effects of the delivered drug, orenhance the activity of the drug or non-therapeutic agent describedabove in the body. In addition, the nanodevice may contain agents thatenhance targeting of the drug or agent to specific regions of the body,including different cell types, tissues, or organs.

II. Administration of Nanodevices

According to the present methods, a the rapeutically effective amount ofthe nanodevice is delivered to a patient. As used herein, “atherapeutically effective amount” of the nanodevice is the quantity ofthe nanodevice required to achieve a desired clinical outcome, such as adecrease in infarct size in a mammalian heart due to an ischemic orother cell damaging event. This amount will vary depending on the timeof administration (e.g., prior to an ischemic event, at the onset of theevent or thereafter), the route of administration, the duration oftreatment, the specific nanodevice used, and the characteristics(including the health) of the patient, as known in the art. The skilledartisan will be able to determine the optimum dosage.

Generally, the concentration or dosage of a nanodevice for use accordingto the present methods is about 100 to 10,000 million particles/mL, butis preferably about 10 to 100,000 million particles/mL. Alternatively,approximately 10 μL to 10 mL, and preferably 100 μL to 1 mL, of an0.0001% to 2.5% aqueous nanodevice suspension, and preferably a 0.00025%to a 0.0025% aqueous suspension, is delivered endovascularly.

The nanodevices are typically administered parenterally, withintravenous administration being preferred. It will be appreciated thatthe nanodevices can include any necessary or desirable pharmaceuticalexcipients to facilitate delivery. Exemplary excipients include, but arenot limited to water, saline, buffers, oils, or other liquids. Thecarrier may be selected for intravenous or intraarterial administration,and may include a sterile aqueous or non-aqueous solution that mayinclude preservatives, bacteriostats, buffers, and/or antioxidants knownto the art.

Administration of the nanodevices can be to any tissue or organ in thebody for protection from ischemic damage or reduction of ischemicdamage. Delivery of nanodevices to protect or reduce damage in theheart, liver, kidney, brain, etc. are contemplated. One skilled in theart will recognize that the described nanoparticles can be used toprepare a medicament for administration to a mammalian patient forreducing cellular damage due to ischemia.

III. Proposed Mechanism

The mechanism of action is not critical to the present methods. However,a proposed mechanisms of action includes, but is not limited to, causingminor damage to cells or tissues associated with a target organ, orperipheral cells or tissues, thereby causing the release of endogenouscytoprotective agents, which confer tissue protection. This method oftissue protection is illustrated in the example below, in particularwith respect to the use of PMM nanoparticles in an animal model forcardiac ischemic and reperfusion injury.

EXAMPLES

Reference will now be made to specific examples illustrating aspects andembodiments described above. The examples are provided to illustratepreferred embodiments and should not be construed as limiting the scopeof the subject matter.

Example 1 Nanodevices for Reducing Ischemic Injury

The present example shows that reduced tissue damage was achieved in arat heart subject to ex vivo ischemia and reperfusion injury by treatingthe heart with nanoparticles.

A. Materials

Male Sprague-Dawley rats (280-380 g; n=4-5 per group) were used for exvivo cardiac ischemia and reperfusion. PMM nanoparticles weresynthesized via polymerization of poly(methylidene malonate 2.1.2monomer as described previously (Breton, P. et al., Biomaterials,19(1-3):271 (1998)). Polymer was delivered at 1×10⁶ particles/mL to theheart.

B. Methods

To mimic acute myocardial infarction, an ex vivo Langendorff cardiacischemia/reperfusion model was used. Working hearts were extracted fromdeeply anesthetized animals and rapidly cannulized. Oxygenated, warmedKrebs buffer was perfused in a retrograde fashion through the hearts viathe aorta to maintain tissue viability. To mimic ischemia, perfusion ofthe oxygenated buffer was stopped for a period of 40 minutes.Reperfusion was performed by recommencing cardiac perfusion of bufferfor a period of 60 minutes. During the reperfusion period, perfusatefrom the heart was collected to measure creatine phosphokinase (CPK)levels. CPK is a molecule released from necrotic cardiomyocytes, whichis used to monitor the extent of cardiac infarction. Followingreperfusion, hearts were sliced and stained with triphenyl tetrazoliumchloride (TTC), a mitochondrial stain that delineates live/dead tissue.Infarct size was quantitated by measuring the area of infarction withrespect to the total heart surface.

Hearts underwent treatment with either sham buffer (control) or a buffercontaining PMM nanoparticles, for 10 minutes prior to ischemia. Thisbuffer was either re-circulated through the heart in a closed-loopsystem or, in different sets of experiments, received fresh buffer in anopen loop system. CPK levels were monitored from heart perfusate duringthe period of reperfusion for additional quantitation of cell damage.

C. Results

Results are shown in FIGS. 1, 2, and 3. In sham-buffer-treated animals,a large area of cardiac infarction developed (FIGS. 1, 2), whichcorresponded to high CPK levels (FIG. 3). When PMM nanoparticles wereperfused through the heart, using recirculation of perfusate backthrough the heart, a substantial reduction in infarct size was achieved(FIGS. 1, 2), corresponding to a decrease in CPK levels (FIG. 3). Thus,delivery of nanodevices to the animals conferred protection from tissuedamage due to ischemia. In contrast, when nanoparticles were notre-circulated through the heart in the perfusate buffer, no significantprotection was afforded (FIGS. 1, 2, 3).

Without being bound by theory, the results suggest that one or moreendogenous releasable factors (e.g., adenosine) is stimulated upondelivery of nanodevices, and such endogenous releasable factors mediatea protective effect. The endogenous factor(s) could be released due tothe interaction of the nanoparticle on the target tissue, and/or mayfurther be due to interaction of the nanoparticles with the vessel wall,causing stress, such as shear-stress, and cellular responses, such asrelease of endogenous vasodilating or cytoprotective agents.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions and sub-combinations as are within their truespirit and scope.

Example 2 Identification of Optimal Surface Characteristics ofNanodevices

Nanodevices having surface characteristic optimal for delivering andreleasing therapeutic agents (i.e., drugs) are determined by testing aseries of nanodevices, e.g., in the animal model described in Example 1.

In one experiment, the following nanodevices are tested.

Nanodevices with differing surface characteristics will be evaluated forcardioprotective effects Surface characteristics Size Name Fluorophore(λ) Negative charge 0.5 μm PMM 2.1.2 580/605 Positive charge 0.2 μm FSamine 580/605 Positive charge 1.0 μm FS amine 505/515 Carboxylatemodified 0.2 μm FS carboxylate 580/605 Carboxylate modified 0.5 μm FScarboxylate 580/605 Carboxylate modified 1.0 μm FS carboxylate 580/605

Each nanodevice is tested as described, e.g., in Example 1, with resultsbeing determined by measuring the levels of cardiac enzymes released inthe buffer effluent (a measure of oncosis) as well as by staining withtriphenyl tetrazolium chloride (TTC) to measure tissue necrosis.

Upon identifying the optimal nanodevice surface for elicitingprotection, experiments are performed to determine if the particularsurface characteristics are required for endothelial interactions.Tissue collected from the above study is cut into 5-micron transversesections via a cryostat, and then mounted and fixed on a glass slide.The sections are co-stained for nuclei (DAPI) and an endothelial marker(CD31). Fluorescent images are obtained using a laser scanning confocalmicroscope (Pascal, Zeiss) with the appropriate excitation/emissionswavelength for the fluorophore, as provided in the above Table.

These experiments determine the optimum surface for eliciting protectionand whether such surfaces facilitate interaction between the nanodevicesand the epithelium of a transplant organ.

Example 3 Measuring Adenosine Concentration in Cardiac Tissue

Preliminary studies suggest that nanodevices protect allografts via anadenosine receptor-dependent mechanism. However, the short half-life ofadenosine (<1 second in the blood) makes studying this mechanismproblematic. An inhibitor of adenosine deaminase (the enzyme responsiblefor degradation of adenosine and used for snap freezing) is used tofacilitate study the mechanism. Briefly, hearts are perfused withcardioprotective nanoparticles for 2 minutes. A cold 15G needle is usedto collect biopsy samples every 30 seconds. The biopsy samples are snapfrozen in tubes containing extraction buffer (0.4 M perchloric acid) and280 μM deaminase inhibitor (e.g., erythro-9(2-hydroxy-3-nonyl)adenine;EHNA. Following two freeze/thaw cycles, shaking, and centrifugation, thesamples are loaded onto a reverse-phase HPLC column to separateadenosine from other nucleotide catabolites. Samples are normalized toan adenosine standard and the relative amount of adenosine is recorded.

The difference in adenosine levels between organs treated with ananodevice and untreated organs indicates the effect of the nanodevicetreatment. For example, elevated levels of adenosine followingnanodevice treatment suggest that the mechanism of action of thenanodevices involves increasing adenosine levels.

Example 4 Determining the Activity of 5′ Nucleotidase

Adenosine is generated from the 5′ nucleotidase-mediated catabolism ofAMP. To determine if the activity of this enzyme is altered by treatmentwith nanodevices, treated and untreated organs (e.g., hearts) areperfused for 2 minutes with nanoparticles. Needle biopsy samples arethen collected, snap-frozen, and ground using a mortar and pestle cooledwith liquid nitrogen. The production of adenosine is monitoredspectrophotometrically at 340 nm upon addition of AMP to biopsymaterial. Specificity of adenine production may be determined byaddition of the 5′nucleotidase inhibitor alpha, beta-methylene adenosine5′-diphosphate (AOPCP), which should prevent the production of adenosineby the subject pathway. Samples may normalized to an adenosine standard.

It is claimed:
 1. A method for reducing ischemic reperfusion injury to atissue, comprising delivering intravascularly to the tissue acomposition comprised of nanodevices.
 2. The method of claim 1, whereinsaid administering comprises administering during organ or tissueharvest.
 3. The method of claim 1, wherein said administering comprisesadministering during organ or tissue transplantation.
 4. The method ofclaim 1, wherein said administering comprises administering duringsurgery.
 5. The method of claim 1, wherein said administering comprisesadministering polymeric nanodevices.
 6. The method of claim 1, whereinsaid administering comprises administering lipidic nanodevices.
 7. Themethod of claim 1, wherein said administering comprises administeringnanodevices further comprising a therapeutic agent.
 8. A method forconferring protection to a tissue at risk of an ischemic reperfusioninjury to a tissue, comprising administering intravascularly to thetissue a composition comprised of polymer or lipidic nanodevices.
 9. Themethod of claim 8, wherein said administering comprising administeringprior to a scheduled ischemic event.
 10. The method of claim 9, whereinsaid scheduled ischemic event is surgery.
 11. The method of claim 10,wherein said administering is at least about 12 hours prior to surgery.12. The method of claim 8, wherein said administering comprisingadministering chronically.
 13. The method of claim 12, wherein saidadministering is at least about monthly.
 14. The method of claim 8,wherein said administering comprising administering lipidic nanodevicesin the form of liposomes or micelles.
 15. The method of claim 8, whereinsaid administering comprising administering nanodevices furthercomprising a therapeutic agent.